The ribosome is the two-subunit macromolecular machine responsible for the decoding of the messenger RNA into the encoded polypeptide - the translation of the genetic code. In fulfilling this essential cellular role, ribosomes carry out a number of discrete functions including selecting the appropriate aminoacyl tRNAs, catalyzing peptide bond formation (PT), accurately translocating the mRNA:tRNA complex and releasing the completed polypeptide chain. Theoretical considerations of the origin of life predict a central role for the rRNAs in translation. This view has been strengthened by the demonstration of catalysis by RNA, extreme conservation observed among rRNA sequences and finally by atomic resolution structures of the ribosome displaying primarily rRNA in the functionally critical regions. The long-term goal of our work is to understand the molecular mechanics of ribosome function. We are particularly interested in understanding how the rRNA and tRNA components of the translation machinery contribute to the overall process of protein synthesis. The advent of atomic resolution structural information and lower resolution information from cryoEM has substantially changed the focus of our research during the past ten years. While the previous challenge was to identify elements of the ribosome located in functionally critical regions, the goal now is to understand how these structurally identified critical components function. The ribosome provides an excellent, tractable system for dissecting molecular movements and examining how they define biologically fundamental signal transduction pathways. All three specific aims are built around pre-steady state kinetic approaches and the analysis of mutated translation components to dissect ribosome function at the molecular level. We are particularly interested in the molecular details of how the ribosome selects the cognate aminoacyl-tRNA on sense codons and the appropriate release factor on stop codons, and how these mechanisms are related. A major new focus of this proposal is the exploration of the mechanistic features of a novel quality control system on the ribosome that takes place following peptide bond formation. PUBLIC HEALTH RELEVANCE: The ribosome is the target of many natural and synthetic antibiotics that affect distinct steps in the translation cycle. Information obtained from detailed molecular studies proposed here will help in defining the features of current antibiotics which make them effective and will aid in the design of more effective therapeutics.